Treatment of neurodegenerative diseases, causation of memory enhancement, and assay for screening compounds for such

ABSTRACT

Methods for enhancing memory and/or learning and prevent neurodegeneration by administration of certain heterocyclic and aromatic compounds are described. The methods are particularly useful for treating patients suffering from a neurodegenerative disease such as (without limitation) Alzheimer&#39;s, Parkinsons&#39;s, and Lou Gehrig&#39;s (ALS) disease or memory or learning impairment. A neuronal human cell-based assay that assess NF-kB gene up-regulation using a luciferase reporter is also provided that screens for compounds useful in methods for enhancing memory or learning.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a Continuation of U.S. patent application Ser. No.13/501,934, filed Jun. 6, 2012. U.S. patent application Ser. No.13/501,934 is a National Stage of International Application No.PCT/US2010/052624, filed on Oct. 14, 2010, and claims priority of U.S.Provisional Patent Application No. 61/251,874, filed on Oct. 15, 2009.The disclosures of the prior applications are incorporated herein intheir entirety by reference.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was partially supported by Grant MLSCN: 1U54-HG-003917and —N01NS-22348 and 1 R03MH082367-01 to M.G. from National Institute ofHealth and the US Government has certain rights in the invention.

TECHNICAL FIELD

The present disclosure relates to a method for treating a patientsuffering from a neurodegenerative disease such as Alzheimer's,Parkinson's, and Lou Gehrig's (ALS) disease, and patients with orpatients predisposed to developing a learning and memory impairmentand/or neurodegeneration not classifiable in any of the above mentionedexamples and enhancing memory performance in normal and pathologicalstates, which comprises administering to the patient an effective amountof certain heterocyclic and aromatic compounds. The applications of thisdisclosure also include all of the situations in which strengtheningNF-kB signaling can result in amelioration of patient conditions and isnot limited to the central nervous system or the above mentioned centralnervous system conditions. A number of the compounds to be employed arenovel. The present disclosure also relates to a neuronal humancell-based assay that will assess NF-kB gene up-regulation using aluciferase reporter for screening for compounds for use in treatingneurodegenerative diseases as described above.

BACKGROUND ART

Effective treatment for neurodegenerative diseases, such as (withoutlimitation) Alzheimer's, Parkinson's, and Lou Gehrig's disease is stilllacking. For example, it has been recently reported that Alzheimer'sdisease is the seventh leading cause of death in the United States. Ithas also been reported that 26 million people worldwide, including 5million Americans, have Alzheimer's disease. Only marginalsymptomatologic treatment is available to date. Statistics andprojections indicate that 1 in 2 subjects above the age of 80 experiencesome level of clinically relevant cognitive impairment and with theprojections indicating an increase of the average lifespan of the humansthe burden deriving to society will be immense.

There are several indications that the NF-kB pathway plays a role inneuronal resilience and in the changes induced by cellular learning suchas long term potentiation and depression. Several reports have shownthat knocking out NF-kB activity in the brain causes sensitization totoxic stimuli, such as β-amyloid, excitatory aminoacids and to trauma.Also NF-kB activation has been involved in long term potentiation anddepression the cellular correlates of learning and memory. In addition,activation of NF-kB is a known anti-apoptosis mechanism. Failure ofNF-kB in other systems can also be counteracted by compounds of thisdisclosure and therefore will be covered by this disclosure.

SUMMARY OF DISCLOSURE

The present disclosure relates to a method for protecting neurons andenhancing memory performance in a patient or for treating a patientsuffering from a neurodengenerative disease a memory impairment, or alearning impairment by administering to the patient at least onecompound represented by the structures:

a pharmaceutically acceptable salt thereof, a solvate thereof, a prodrugthereof and mixtures thereof; in an amount effective for treating saidpatient.

In Structure 1, Z represents O, NH, N—R₃, S, CH, or CR₃; and each R₁, R₂and R₃ is individually selected from the group consisting of substitutedor unsubstituted alkyl, aryl, aralkyl and heteroaryl.

In Structure 1a, Z represents N, CH, or CR₃ and each R₁, R₂ and R₃ isindividually selected from the group consisting of substituted orunsubstituted alkyl, aryl, aralkyl and heteroaryl.

In Structure 2, Y is O or S; R₁ is H, acyl, substituted or unsubstitutedalkyl, aryl, aralkyl and heteroaryl; each R₂ and R₄ is individuallyselected from the group consisting of substituted or unsubstitutedalkyl, aryl, aralkyl and heteroaryl and R₃ is selected from the groupconsisting of H or substituted or unsubstituted alkyl, aryl, aralkyl andheteroaryl.

In Structure 3, X represents OR₆ or NR₆R₇; each R₁ and R₂ isindividually selected from the group consisting of single or multiplesubstitutions of H or substituted or unsubstituted alkyl, aryl, aralkylheteroaryl or acyl, halogen, hydroxy, alkoxy, amino or substitutedamino; each R₃, R₄ and R₆ is individually selected from the groupconsisting of H, substituted or unsubstituted alkyl, aryl, aralkyl andheteroaryl; and each R₅ and R₇ is individually selected from the groupconsisting of H, acyl, substituted or unsubstituted alkyl, aryl, aralkyland heteroaryl.

In Structure 4, each W, X, Y, Z is N or CR₆; each R₁, R₂, R₄, R₅ and R₆is individually selected from the group consisting of H, substituted orunsubstituted alkyl, aryl, aralkyl and heteroaryl; R₃ is H, acyl,substituted or unsubstituted alkyl, aryl, aralkyl and heteroaryl.

In Structure 5, each R₁, R₂, R₃ and R₄ is individually selected from thegroup consisting of H, substituted or unsubstituted alkyl, aryl, aralkyland heteroaryl.

In Structure 5a, X represents N or CR₂; Y represents S or CR₅R_(6;) andeach R₁, R₂, R₃, R₄, R₅ and R₆ is individually selected from the groupconsisting of H, substituted or unsubstituted alkyl, aryl, aralkyl andheteroaryl.

In Structure 6, X represents R₂ or NR₃R₄; Y represents O, S or NR₅; R₁is selected from the group consisting of single or multiplesubstitutions of H or substituted or unsubstituted alkyl, aryl, aralkylheteroaryl or acyl, halogen, hydroxy, alkoxy, amino or substitutedamino; each R₂, R₃, and R₅ is individually selected from the groupconsisting of H, substituted or unsubstituted alkyl, aryl, aralkyl andheteroaryl; and R₄ is H, acyl, substituted or unsubstituted alkyl, aryl,aralkyl and heteroaryl.

In Structure 7, X represents O—R₄ or NR₄R₅; each R₁, R₂, R₃, and R₄ isindividually selected from the group consisting of H, substituted orunsubstituted alkyl, aryl, aralkyl and heteroaryl; and R₅ is H, acyl,substituted or unsubstituted alkyl, aryl, aralkyl and heteroaryl.

In Structure 8, X represents O or S; Y represents N or CR₃; Z representsNR₄R₆ or CR₄R₅R₆; each R₃, R₄, and R₆ is individually selected from thegroup consisting of H, substituted or unsubstituted alkyl, aryl, aralkyland heteroaryl; and n is o, 1, 2, 3, or 4.

In Structure 9, X represents O, S or NR₄; R₁ is selected from the groupconsisting of single or multiple substitutions of H or substituted orunsubstituted alkyl, aryl, aralkyl heteroaryl or acyl, halogen, hydroxy,alkoxy, amino or substituted amino; and each R₂, R₃, and R₄ isindividually selected from the group consisting of H, substituted orunsubstituted alkyl, aryl, aralkyl and heteroaryl.

In Structure 10, R₁ is selected from the group consisting of single ormultiple substitutions of H or substituted or unsubstituted alkyl, aryl,aralkyl heteroaryl or acyl, halogen, hydroxy, alkoxy, amino orsubstituted amino; R₂ is selected from the group consisting of H,substituted or unsubstituted alkyl, aryl, aralkyl and heteroaryl; R₃ isH, acyl, substituted or unsubstituted alkyl, aryl, aralkyl andheteroaryl; m is 0, 1, 2, or 3; and n is 1, 2, or 3.

In Structure 10a, R₁ is selected from the group consisting of single ormultiple substitutions of H or substituted or unsubstituted alkyl, aryl,aralkyl heteroaryl or acyl, halogen, hydroxy, alkoxy, amino orsubstituted amino; R₄ is selected from the group consisting of H,substituted or unsubstituted alkyl, aryl, aralkyl and heteroaryl; R₃ isH, acyl, substituted or unsubstituted alkyl, aryl, aralkyl andheteroaryl; and m is 0, 1, 2, or 3.

In Structures 11 and 12 R₁, is selected from the group consisting ofsingle or multiple substitutions of H or substituted or unsubstitutedalkyl, aryl, aralkyl heteroaryl or acyl, halogen, hydroxy, alkoxy, aminoor substituted amino; R₂ is selected from the group consisting of H,substituted or unsubstituted alkyl, aryl, aralkyl and heteroaryl; and R₃is H, acyl, substituted or unsubstituted alkyl, aryl, aralkyl andheteroaryl.

In Structure 13, each R₁ and R₂ is individually selected from the groupconsisting of single or multiple substitutions of H or substituted orunsubstituted alkyl, aryl, aralkyl heteroaryl or acyl, halogen, hydroxy,alkoxy, amino or substituted amino.

In Structure 14, R₁ is selected from the group consisting of single ormultiple substitutions of H or substituted or unsubstituted alkyl, aryl,aralkyl heteroaryl or acyl, halogen, hydroxy, alkoxy, amino orsubstituted amino; and each R₂ and R₃ is individually selected from thegroup consisting of H or substituted or unsubstituted alkyl, aryl,aralkyl heteroaryl or acyl, halogen, hydroxy, alkoxy, amino, substitutedamino, alkylthio, cyano or azido.

In Structure 15, R₁ is selected from the group consisting of single ormultiple substitutions of H or substituted or unsubstituted alkyl, aryl,aralkyl heteroaryl or acyl, halogen, hydroxy, alkoxy, amino orsubstituted amino, cyano or alkylthio; and each R₂ and R₃ isindividually selected from the group consisting of H or substituted orunsubstituted alkyl, aryl, aralkyl heteroaryl or acyl, halogen, hydroxy,alkoxy, amino, substituted amino, cyano or alkylthio.

In Structure 16, each R₁ and R₂ is individually selected from the groupconsisting of single or multiple substitutions of H or substituted orunsubstituted alkyl, aryl, aralkyl heteroaryl or acyl, halogen, hydroxy,alkoxy, amino or substituted amino, cyano, azido or alkylthio.

The present disclosure also relates to treating a patient that ispredisposed to developing a neurodegenerative disease, a memoryimpairment, or a learning impairment.

In another embodiment, the method is for improving learning. In yetanother embodiment, the method is for preventing or minimizing thedecline of memory or improving or maintaining baseline memory.

The present disclosure also relates to novel compounds employedaccording to this disclosure.

The present disclosure also relates to a neuronal human cell-based assaythat will assess NF-kB up-regulation using a luciferase reporter forscreening for compounds that can be used in treating neurodegenerativediseases.

Still other objects and advantages of the present disclosure will becomereadily apparent by those skilled in the art from the following detaileddescription, wherein it is shown and described preferred embodiments,simply by way of illustration of the best mode contemplated. As will berealized the disclosure is capable of other and different embodiments,and its several details are capable of modifications in various obviousrespects, without departing from the disclosure. Accordingly, thedescription is to be regarded as illustrative in nature and not asrestrictive.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a schematic of the construct used in this disclosure. A TATAbox has been attached to 4 copies of the NF-kB promoter enhancersequence to drive firefly luciferase gene transcription.

FIG. 2 shows SH-5YSY cells that were exposed to increasingconcentrations of blasticidin.

FIG. 3 shows the effect of TNF at 5 and 10 nM on the expression of thefirefly lucipherase in different selected clones. Stimulation with TNFshowed several high expressing clones. The clone C1 was the strongestexpresser and was selected for further analysis.

FIG. 4 shows the effect of different cell numbers and two TNF-αconcentrations on luciferase expression in the clone C1.

FIG. 5 shows a comparison between phenol red free HEPES-buffered DMEMand phenol red containing bicarbonate-buffered DMEM in C1 clone.

FIG. 6 is a graph illustrating the concentration-dependent effect ofDMSO on TNF-induced lucipherase and on cell numbers in C1 clone.

FIG. 7 is a graph showing the effect of increasing concentration of DMSOon the survival of the C1 clone cells. Cells were exposed to DMSO for 24hours as they would in a screening run.

FIG. 8 is a graph showing the concentration dependent effect of TNF-α onNF-kB promoter driven luciferase expression.

FIG. 9 gives examples of Z′ plate arrays and results. In panel A cellswere laid out in a plate and then exposed to vehicle or to 5 ng/ml TNFfor 24 hours. Treatments were performed in ¼ quadrant with a crosspattern. In these settings Z′ values above 0.7 have consistently beenperformed. In panel B instead of a quarter arrays, control and TNFtreated cells were scattered across the plate. Even in this randompattern Z′ values were consistently above 0.7.

FIG. 10 is a collection of Z′ values determined on a robotic platformduring a screening campaign; Z′ values were always consistent with avery robust assay.

FIG. 11 shows structures of specific compounds selected for furtherfollow up testing.

FIG. 12 correlates structures from FIG. 11 with internal SRIdesignations.

FIG. 13 shows the activation of NF-kB p65 in primary neurons. Nucleartranslocation/activation of NF-kB p65 in response to the indicated CMPDsafter 24 h exposure is shown and compared to control and TNF-α (24hours) treated cells. Examples of nuclear p65 are highlighted by thearrows. Image analysis allowed us to quantify the data and performstatistical analysis. A significant increase of nuclear presence of p65is shown in the bar graphs in panels C4, D4 and E4. Panel F4 shows theneuroprotective effect of CMPD 22782 as a prototype compound.

FIG. 14 shows the effect of prototype CMPDs on I-kB and NF-kB p55. A)I-kB was not affected by CMPD exposure while it was reduced by TNF. B)CMPDs increased cytoplasmic NF-kB p65. C) CMPDs increased nuclearlocalization of NF-kB p65. D) The sum of CMPD effects on P65 in thecytoplasm and in the nucleus.

FIG. 15 shows the activation of NF-kB p65 in primary neurons by neuronselective CMPDs. Nuclear translocation of NF-kB p65 in response to CMPDsafter 24 h exposure is shown in and compared to control and 100 ng/mlTNF-α (15 min) treated cells. Examples of nuclear p65 are highlighted bythe arrows. Image analysis allowed quantification of the data andstatistical analysis. A significant increase larger or equal to thestrong effect of TNF of nuclear p65 is shown in panels E and F for theCMPDs.

FIG. 16 is a four graph collection showing the effect of 4 of thecompounds included in this document as prototype on the induction ofMnSOD a neuroprotective enzyme that is actuated by NF-kB induction.

FIG. 17 (A-C) shows the neuroprotective effect of prototype compoundsaccording to the present disclosure on three well established in vitromodels of neurodegeneration that are widely use to test in vitroeffective compounds.

FIG. 18 shows the effect of the indicated compound on the toxic effectof glutamate in primary neurons. The data indicate that 60% of the celldeath induced by glutamate is prevented by the compound 22872.

FIG. 19 shows the effect of CMPD 22819 on neurotoxicity induced by H₂O₂.Primary rat cortical neurons at 6 D.I.V. were pretreated for 24 hourswith 300 nM of 22819 and exposed to toxicity by 120 μM of H₂O₂. Neuronspretreated with the CMPD showed a 62% reduction of the H₂O₂ inducedtoxicity.

FIG. 20 (A-B) shows the effect of CMPD on MnSOD activity in primaryneurons. A) CMPDs active in astrocytes increased activity of MnSODactivity in primary neurons. B) CMPDs inactive in astrocytes alsoincreased NF-kB driven MnSOD expression\activity in neurons.

FIG. 21 shows the in silico predicted parameters for the braindistribution of 7 neuron selective compounds. ADMET BBB is the log ofbrain to blood partition coefficient calculated using the softwarePipeline Plot. ADMET BBB Level indicates the ranking of ADMET BBB (0being very high predicted passive distribution in the brain decreasingdown to 3. 4 indicated unpredictable behavior). QPLog Predictedbrain/blood partition coefficient (Range=−3.0 to 1.2 higher the better)using the software Quik Prop. CNS: Predicted central nervous systemactivity on a −2 (inactive) to +2 (active) scale. QPPMDCK: Predictedapparent MDCK cell permeability in nm/sec (v<25 poor; v>500 good).QPlogPo/w: Predicted octanol/water partition coefficient (Range=−2.0 to6, optimal v>1 and V<4). QPlogPo/w<5 (Lipophilicity); donorHB <=5(Hydrogen bond donors); accptHB <=10 (Hydrogen) Rule Of Five: Number ofviolations of Lipinski's rule of five (Desired values should be: MW<500(Molecular Weight); bond acceptors.

FIG. 22 (A-D) shows the effect of treatment with SRI22818 and 22819 ondevelopment of ALS-like symptoms in G93A mice. A) Shows that survivalwas significantly improved in animals treated with both CMPDs; B) showsthat reaching the 50% death threshold was significantly delayed; C)shows that the onset of the symptoms is slightly affected, whilstprogression through the 4 neurological grades was greatly delayed; andD) shows that the weight loss due to muscle atrophy is alsosignificantly delayed in animals treated with the two CMPDs.

DETAILED DESCRIPTION OF THE DISCLOSURE

In particular, the present disclosure relates to use of compoundsrepresented by the following structures:

a pharmaceutically acceptable salt thereof, a solvate thereof, a prodrugthereof and mixtures thereof.

In Structure 1, Z represents O, NH, N—R₃, S, CH, or CR₃; and each R₁, R₂and R₃ is individually selected from the group consisting of substitutedor unsubstituted alkyl, aryl, aralkyl and heteroaryl.

In Structure 1a, Z represents N, CH, or CR₃ and each R₁, R₂ and R₃ isindividually selected from the group consisting of substituted orunsubstituted alkyl, aryl, aralkyl and heteroaryl.

In Structure 2, Y is O or S; R₁ is H, acyl, substituted or unsubstitutedalkyl, aryl, aralkyl and heteroaryl; each R₂ and R₄ is individuallyselected from the group consisting of substituted or unsubstitutedalkyl, aryl, aralkyl and heteroaryl and R₃ is selected from the groupconsisting of H or substituted or unsubstituted alkyl, aryl, aralkyl andheteroaryl.

In Structure 3, X represents OR₆ or NR₆R₇; each R₁ and R₂ isindividually selected from the group consisting of single or multiplesubstitutions of H or substituted or unsubstituted alkyl, aryl, aralkylheteroaryl or acyl, halogen, hydroxy, alkoxy, amino or substitutedamino; each R₃, R₄ and R₆ is individually selected from the groupconsisting of H, substituted or unsubstituted alkyl, aryl, aralkyl andheteroaryl; and each R₅ and R₇ is individually selected from the groupconsisting of H, acyl, substituted or unsubstituted alkyl, aryl, aralkyland heteroaryl.

In Structure 4, each W, X, Y, Z is N or CR₆; each R₁, R₂, R₄, R₅ and R₆is individually selected from the group consisting of H, substituted orunsubstituted alkyl, aryl, aralkyl and heteroaryl; R₃ is H, acyl,substituted or unsubstituted alkyl, aryl, aralkyl and heteroaryl.

In Structure 5, each R₁, R₂, R₃ and R₄ is individually selected from thegroup consisting of H, substituted or unsubstituted alkyl, aryl, aralkyland heteroaryl.

In Structure 5a, X represents N or CR₂; Y represents S or CR₅R_(6;) andeach R₁, R₂, R₃, R₄, R₅ and R₆ is individually selected from the groupconsisting of H, substituted or unsubstituted alkyl, aryl, aralkyl andheteroaryl.

In Structure 6, X represents R₂ or NR₃R₄; Y represents O, S or NR₅; R₁is selected from the group consisting of single or multiplesubstitutions of H or substituted or unsubstituted alkyl, aryl, aralkylheteroaryl or acyl, halogen, hydroxy, alkoxy, amino or substitutedamino; each R₂, R₃, and R₅ is individually selected from the groupconsisting of H, substituted or unsubstituted alkyl, aryl, aralkyl andheteroaryl; and R₄ is H, acyl, substituted or unsubstituted alkyl, aryl,aralkyl and heteroaryl.

In Structure 7, X represents O—R₄ or NR₄R₅; each R₁, R₂, R₃, and R₄ isindividually selected from the group consisting of H, substituted orunsubstituted alkyl, aryl, aralkyl and heteroaryl; and R₅ is H, acyl,substituted or unsubstituted alkyl, aryl, aralkyl and heteroaryl.

In Structure 8, X represents O or S; Y represents N or CR₃; Z representsNR₄R₅ or CR₄R₅R₆; each R₃, R₄, and R₆ is individually selected from thegroup consisting of H, substituted or unsubstituted alkyl, aryl, aralkyland heteroaryl; and n is o, 1, 2, 3, or 4.

In Structure 9, X represents O, S or NR₄; R₁ is selected from the groupconsisting of single or multiple substitutions of H or substituted orunsubstituted alkyl, aryl, aralkyl heteroaryl or acyl, halogen, hydroxy,alkoxy, amino or substituted amino; and each R₂, R₃, and R₄ isindividually selected from the group consisting of H, substituted orunsubstituted alkyl, aryl, aralkyl and heteroaryl.

In Structure 10, R₁ is selected from the group consisting of single ormultiple substitutions of H or substituted or unsubstituted alkyl, aryl,aralkyl heteroaryl or acyl, halogen, hydroxy, alkoxy, amino orsubstituted amino; R₂ is selected from the group consisting of H,substituted or unsubstituted alkyl, aryl, aralkyl and heteroaryl; R₃ isH, acyl, substituted or unsubstituted alkyl, aryl, aralkyl andheteroaryl; m is 0, 1, 2, or 3; and n is 1, 2, or 3.

In Structure 10a, R₁, is selected from the group consisting of single ormultiple substitutions of H or substituted or unsubstituted alkyl, aryl,aralkyl heteroaryl or acyl, halogen, hydroxy, alkoxy, amino orsubstituted amino; R₄ is selected from the group consisting of H,substituted or unsubstituted alkyl, aryl, aralkyl and heteroaryl; R₃ isH, acyl, substituted or unsubstituted alkyl, aryl, aralkyl andheteroaryl; and m is 0, 1, 2, or 3.

In Structures 11 and 12, R₁ is selected from the group consisting ofsingle or multiple substitutions of H or substituted or unsubstitutedalkyl, aryl, aralkyl heteroaryl or acyl, halogen, hydroxy, alkoxy, aminoor substituted amino; R₂ is selected from the group consisting of H,substituted or unsubstituted alkyl, aryl, aralkyl and heteroaryl; and R₃is H, acyl, substituted or unsubstituted alkyl, aryl, aralkyl andheteroaryl.

In Structure 13, each R₁ and R₂ is individually selected from the groupconsisting of single or multiple substitutions of H or substituted orunsubstituted alkyl, aryl, aralkyl heteroaryl or acyl, halogen, hydroxy,alkoxy, amino or substituted amino.

In Structure 14, R₁ is selected from the group consisting of single ormultiple substitutions of H or substituted or unsubstituted alkyl, aryl,aralkyl heteroaryl or acyl, halogen, hydroxy, alkoxy, amino orsubstituted amino; and each R₂ and R₃ is individually selected from thegroup consisting of H or substituted or unsubstituted alkyl, aryl,aralkyl heteroaryl or acyl, halogen, hydroxy, alkoxy, amino, substitutedamino, alkylthio, cyano or azido.

In Structure 15, R₁ is selected from the group consisting of single ormultiple substitutions of H or substituted or unsubstituted alkyl, aryl,aralkyl heteroaryl or acyl, halogen, hydroxy, alkoxy, amino orsubstituted amino, cyano or alkylthio; and each R₂ and R₃ isindividually selected from the group consisting of H or substituted orunsubstituted alkyl, aryl, aralkyl heteroaryl or acyl, halogen, hydroxy,alkoxy, amino, substituted amino, cyano or alkylthio.

In Structure 16, each R₁ and R₂ is individually selected from the groupconsisting of single or multiple substitutions of H or substituted orunsubstituted alkyl, aryl, aralkyl heteroaryl or acyl, halogen, hydroxy,alkoxy, amino or substituted amino, cyano, azido or alkylthio.

Listed below are definitions of various terms used to describe thisinvention. These definitions apply to the terms as they are usedthroughout this specification, unless otherwise limited in specificinstances, either individually or as part of a larger group.

Typical aliphatic acyl groups contain 1 to 6 carbon atoms and includeformyl, acetyl, propionyl and isobutyryl.

Typical aromatic acyl groups include unsubstituted and alkyl substitutedaromatic groups containing 7 to 10 carbon atoms in the aromatic ring.When substituted the alkyl group typically contains 1-6 carbon atoms.Typical aromatic acyl groups include benzoyl para-toluoyl andphenylacetyl.

The term “alkyl” refers to saturated or unsaturated (alkenyl or alkynyl)straight, branched chain, or cyclic, unsubstituted hydrocarbon groups oftypically 1 to 22 carbon atoms, more typically 1 to 8 carbon atoms, andeven more typically 1 to 4 carbon atoms.

Examples of suitable alkyl groups include methyl, ethyl and propyl.Examples of branched alkyl groups include isopropyl and t-butyl.Examples of cyclic alkyl groups include cyclohexyl andcyclopropylmethyl. Examples of unsaturated alkyl groups include ethynyl,cyclopentenyl, and allyl. Examples of substituted alkyl groups include2-methoxyethyl, 2,2,2-trifluoroethyl, and 2-diethylaminocyclopentenyl.Suitable monoalkylamino groups for X contain 1-6 carbon atoms andinclude monomethylamino, monoethylamino, mono-isopropylamino,mono-n-propylamino, mono-isobutyl-amino, mono-n-butylamino,mono-n-hexylamino, monophenethylamino, or mono-2-pyridylamino. The alkylmoiety can be straight, branched, or cyclic chain.

Suitable dialkylamino groups typically contain 1-6 carbon atoms in eachalkyl group. The alkyl groups can be the same or different and can bestraight, branched or cyclic chain. Examples of some suitable groups aredimethylamino, diethylamino, ethylmethylamino, dipropylamino,dibutylamino, dipentylamino, dihexylamino, methylpentylamino,ethylpropylamino and ethylhexylamino.

Examples of halo groups are Cl, F, Br and I.

The term “aryl” refers to monocyclic or polycyclic aromatic hydrocarbongroups having 6 to 14 carbon atoms in the ring portion, such as phenyl,naphthyl, biphenyl, and diphenyl groups, each of which may besubstituted such as with a halo or alkyl group.

The term “aralkyl” or “alkylaryl” refers to an aryl group bondeddirectly through an alkyl group, such as benzyl or phenethyl.

The term “heteroaryl”, refers to an optionally substituted, unsaturatedaromatic cyclic group, for example, which is a 5 to 7 memberedmonocyclic, 7 to 11 membered bicyclic, or 10 to 15 membered tricyclicring system, which has at least one heteroatom and at least one carbonatom in the ring. Each ring of the heterocyclic group containing aheteroatom may have 1, 2 or 3 heteroatoms selected from nitrogen atoms,oxygen atoms and sulfur atoms, where the nitrogen and sulfur heteroatomsmay also optionally be oxidized and the nitrogen heteroatoms may alsooptionally be quaternized. Examples of heteroaryl groups are pyridyl,imidazolyl, oxazolyl, thiazolyl, isothiazolyl, furyl, thienyl andindolyl.

When substituted, the above groups are typically substituted with ahalo, alkyl, alkoxy or amino group.

It is of course understood that the compounds of the present disclosurerelate to all optical isomers and stereo-isomers at the various possibleatoms of the molecule, unless specified otherwise.

The compounds according to this disclosure may form prodrugs at hydroxylor amino functionalities using alkoxy, amino acids, etc. groups as theprodrug forming moieties. For instance, the hydroxymethyl position mayform mono-, di- or triphosphates and again these phosphates can formprodrugs. For example, see Meier, CycloSal Phosphates as Chemical TrojanHorses for Intracellular Nucleotide Glycosyl-MonophosphateDelivery—Chemistry Meets Biology, European Journal of Organic Chemistry(2006), (5), 1081-1102, Wiley-VCH Verlag GmbH & Co. KGaA, ChemicalAbstracts 144:391234; Drontle et al, Designing a PronucleotideStratagem: Lessons from Amino Acid Phosphoramidates of Anticancer andAntiviral Pyrimidines, Mini-Reviews in Medicinal Chemistry (2004), 4(4),409-419, Bentham Science Publishers Ltd., Chemical Abstracts 141:230392;Cahard et al, Aryloxy Phosphoramidate Triesters as Protides, inMedicinal Chemistry (2004), 4(4), 371-381, Bentham Science PublishersLtd., Chemical Abstracts, 141:218130 and Meier,CycloSal-Pronucleotides-Design of the Concept, Chemistry, and Antiviralactivity, Advances in Antiviral Drug Design (2004), 4, 147-213, ElsevierB.V, Chemical Abstracts 141:133365.

Preparations of such prodrug derivatives are discussed in variousliterature sources (examples are: Alexander et al., J. Med. Chem. 1988,31, 318; Aligas-Martin et al., PCT WO pp/41531, p. 30). The nitrogenfunction converted in preparing these derivatives is one (or more) ofthe nitrogen atoms of a compound of the disclosure.

“Pharmaceutically acceptable salts” refer to derivatives of thedisclosed compounds wherein the parent compound is modified by makingacid or base salts thereof. The compounds of this disclosure form acidand base addition salts with a wide variety of organic and inorganicacids and bases and includes the physiologically acceptable salts whichare often used in pharmaceutical chemistry. Such salts are also part ofthis disclosure. Typical inorganic acids used to form such salts includehydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric,hypophosphoric and the like. Salts derived from organic acids, such asaliphatic mono and dicarboxylic acids, phenyl substituted alkonic acids,hydroxyalkanoic and hydroxyalkandioic acids, aromatic acids, aliphaticand aromatic sulfonic acids, may also be used. Such pharmaceuticallyacceptable salts thus include acetate, phenylacetate, trifluoroacetate,acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, methyl benzoate, o-acetoxybenzoate,naphthalene-2-benzoate, bromide, isobutyrate, phenyl butyrate,β-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate, cabrate,caprylate, chloride, cinnamate, citrate, formate, fumarate, glycollate,heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate,malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate,oxalate, phthalate, teraphthalate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate,propionate, phenylpropionate, salicylate, sebacate, succinate, suberate,sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate,benzene-sulfonate, p-bromobenzenesulfonate, chlorobenzenesulfonate,ethanesulfonate, 2-hydroxyethanesulfonate, methanesulfonate,naphthalene-1-sulfonate, naphthalene-2-sulfonate, p-toleunesulfonate,xylenesulfonate, tartarate, and the like.

Bases commonly used for formation of salts include ammonium hydroxideand alkali and alkaline earth metal hydroxides, carbonates, as well asaliphatic and primary, secondary and tertiary amines, aliphaticdiamines. Bases especially useful in the preparation of addition saltsinclude sodium hydroxide, potassium hydroxide, ammonium hydroxide,potassium carbonate, methylamine, diethylamine, and ethylene diamine.

“Solvates” refers to the compound formed by the interaction of a solventand a solute and includes hydrates. Solvates are usually crystallinesolid adducts containing solvent molecules within the crystal structure,in either stoichiometric or nonstoichiometric proportions.

Many of the compounds employed according to the present disclosure areavailable commercially. Those compounds to be employed in the presentdisclosure that are novel can be made by those of ordinary skill in theart once aware of the present disclosure without undue experimentationby methods available in the art.

For instance with respect to compounds of Structure 1, see Yale et al.,3,5-Disubstituted-1,2,4-oxadiazoles and4,5-dihydro-3,5-disubstituted-1,2,4-oxadiazoles; Journal of HeterocyclicChemistry (1978), 15(8), 1373-8.

With respect to compounds of Structure 2, see Mane et al, Synthesis of2-aryl-3-[p-(2′-substituted-aminothiazol-4′-yl)phenyl]-4-thiazolidinones,Indian Journal of Chemistry, Section B: Organic Chemistry IncludingMedicinal Chemistry (1983), 22B(1), 81-2; Pathak et al., Synthesis ofsome fluoroarylthiazoles and related compounds as potential fungicides,Bokin Bobai (1981), 9(10), 477-80 and Maziere et al., Fluoroarylderivatives of some heterocyclic compounds, Bulletin de la SocieteChimique de France (1963) 1000-3.

With respect to compounds of Structure 3, see Hekimi, WO 2008/014602entitled Preparation of quinoline derivatives as active CLK-1inhibitors. With respect to compounds of Structure 4, see Bowman et al.,Protein Flexibility and Species Specificity in Structure-Based DrugDiscovery: Dihydrofolate Reductase as a Test System, Journal of theAmerican Chemical Society (2007), 129(12), 3634-3640; Sutherland et al.,Three-dimensional quantitative structure-activity andstructure-selectivity relationships of dihydrofolate reductaseinhibitors, Journal of Computer-Aided Molecular Design (2004), 18(5),309-331, Kluwer Academic Publishers; Debnath, Pharmacophore Mapping of aSeries of 2,4-Diamino-5-deazapteridine Inhibitors of Mycobacterium aviumComplex Dihydrofolate Reductase, Journal of Medicinal Chemistry (2002),45(1), 41-53, American Chemical Society; Suling et al.,Antimycobacterial activities of 2,4-diamino-5-deazapteridine derivativesand effects on mycobacterial dihydrofolate reductase, AntimicrobialAgents and Chemotherapy (2000), 44(10), 2784-2793, American Society forMicrobiology; Piper et al., Lipophilic antifolates as agents againstopportunistic infections. 1. Agents superior to trimetrexate andpiritrexim against Toxoplasma gondii and Pneumocystis carinii in invitro evaluations, Journal of Medicinal Chemistry (1996), 39(6),1271-80, American Chemical Society.

With respect to compounds of Structure 5, see Ashwell et al. WO2006/044869 entitled Preparation of pyrimidinyl imidazooxazoles andimidazothiazoles as inhibitors of p38 MAP kinase; Aggarwal et al.,Hypervalent iodine in the synthesis of bridgehead heterocycles. A facileroute to the synthesis of 6-arylimidazo[2,1-b]thiazoles using[hydroxy(tosyloxy)iodo]benzene, Synthetic Communications (2006), 36(7),875-879; Ashwell et al., WO 2004110990 entitled Preparation ofpyrimidinyl imidazothiazoles and imidazooxazoles as inhibitors of p38;O'Daly et al., Electrophilic substitution of imidazo[2,1-b]thiazoles,Journal of the Chemical Society, Perkin Transactions 1: Organic andBio-Organic Chemistry (1972-1999) (1991), (4), 855-60; Meakins et al.,Substituted imidazo[2,1-b]thiazoles from 2-aminothiazoles and α-bromoketones: efficient preparation and proof of structure, Journal of theChemical Society, Perkin Transactions 1: Organic and Bio-OrganicChemistry (1972-1999) (1989), (3), 643-8; Hoffmann et al.,Tetramethoxyethylene. III; Chemische Berichte (1966), 99(6), 1899-1905;and Buu-Hoi, Reaction of ω-bromoacetophenones with 2-aminothiazole and2-aminobenzothiazoles, Bulletin de la Societe Chimique de France (1966),(4), 1277-9.

With respect to compounds of Structure 6, see Wells et al.,4-Substituted 4-Hydroxycyclohexa-2,5-dien-1-ones with SelectiveActivities against Colon and Renal Cancer Cell Lines, Journal ofMedicinal Chemistry (2003), 46(4), 532-541, American Chemical Society;and Stevens et al. WO 2003/004479 entitled Preparation of 4-arylquinolsand analogs thereof as antiproliferative agents, anticancer agents,antimycobacterial agents, antituberculosis agents, and/orthioredoxin/thioredoxin reductase inhibitors.

With respect to compounds of Structure 7, see Botting et al., WO2004/069774 entitled Synthesis of 13C-labeled estrogen analogs;Bondarenko et al., Synthesis of Analogs of Natural IsoflavonoidsContaining Phloroglucinol, Chemistry of Natural Compounds (Translationof Khimiya Prirodnykh Soedinenii) (2003), 39(3), 271-275, KluwerAcademic/Consultants Bureau; Liu et al, Journal of HeterocyclicChemistry (1991), 28(6), 1641-2; and Pivovarenko et al., Synthesis of5,7-dihydroxyisoflavones and their heterocyclic analogs usingacetoformic anhydride, Dopovidi Akademii Nauk Ukrains'koi RSR, Seriya B:Geologichni, Khimichni to Biologichni Nauki (1985), (7), 44-7.

With respect to compounds of Structure 8, see Gorishnii et al.,Synthesis and properties of rhodanine carboxamides, FarmatsevtichniiZhurnal (Kiev) (2001), (2), 64-67; and Gorishnyi et al., Synthesis andantiphlogistic activity of 5-arylidenerhodanin-3-alkanoic acid amides,Farmatsevtichnii Zhurnal (Kiev) (1995), (4), 50-53.

With respect to compounds of Structure 9, see Vettel et al., DE 10039748entitled Production of 3-oxobenzo[b]thiophene methine dyes; Kucharczyket al., Sodium borohydride reduction of 2,3-dihydrothianaphthen-3-ones,Collection of Czechoslovak Chemical Communications (1968), 33(1), 92-9;Treibs, Pyrrole chemistry, Rev. Chim., Acad. Rep. Populaire Roumaine(1962), 7(2), 1345-66, Kucharczyk et al., Improved preparative methodfor thianaphthene and its 2-substituted derivatives, Chemistry &Industry (London, United Kingdom) (1964), (23), 976; Tsekhanskii,Absorption spectra of the nitrobenzamide derivatives of4-aminodiphenylmethane and 4-amino-4′-dimethylaminodiphenyl-methane,lzvestiya Vysshikh Uchebnykh Zavedenii, Khimiya i KhimicheskayaTekhnologiya (1963), 6(2), 252-6; Hallgas, Comparison of measured andcalculated lipophilicity of substituted aurones and related compounds,Journal of Chromatography, B: Analytical Technologies in the Biomedicaland Life Sciences (2004), 801(2), 229-235, Elsevier B.V.

With respect to compounds of Structure 10, see Hedrich et al., U.S. Pat.No. 4,428,881 entitled Control of unwanted vegetation withN-carbamylindolines; and Tachdjian et al., US 2006045953 entitledAromatic amides and ureas and their uses as sweet and/or umami flavormodifiers, tastants and taste enhancers. With respect to compounds ofStructure 11, see Otten et al., The reaction of α-amino-substituteddiphenylphosphine oxide anions with elemental sulfur and selenium. A newroute to thio- and selenoamides, Recueil des Travaux Chimiques desPays-Bas (1994), 113(11), 499-506, Elsevier; Haynes et al., Newchemosterilants for boll weevils, U. S., Agric. Res. Serv., South. Reg.,[Rep.] (1976), ARS-S-131, 30 pp.; Sullivan et al., U.S. Pat. No.2,875,202 entitled Thiofuramides; and Naylor et al., U.S. Pat. No.2,723,969 entitled Neoprene vulcanization accelerators.

With respect to compounds of Structure 12, see Fischer, Vinylogous acylcompounds. XIX. Vinylogous acyl group migration in 2-aminophenol. Acontribution to the isomerization mechanism of mixed diacyl derivativesof 2-aminophenol, Journal fuer Praktische Chemie (Leipzig) (1980),322(1), 99-124. With respect to compounds of Structure 13, see Dossetteret al., WO 2002066477 entitled Preparation of substitutedimidazopyridines for antagonizing gonadotropin releasing hormoneactivity; Bravi et al. WO 2007039146 entitled Preparation of4-carboxypyrazoles as antivirals for treatment of hepatitis C virus(HCV) infection; Godovikova et al., Orientation of bromination reactionof 2-aryl(alkyl)pyrimidazoles, lzvestiya Akademii Nauk SSSR, SeriyaKhimicheskaya (1965), (8), 1434-41; and Buu-Hoi et al., Thiopheneseries. III. Indoles, naphthindoles, pyrrocolines, and pyrimidazolesderived from the thiophene nucleus, Recueil des Travaux Chimiques desPays-Bas et de la Belgique (1949), 68, 441-72.

With respect to compounds of Structure 15, see Deepthi et al., Microwaveinduced dry media DDQ oxidation—a one step synthesis of2-arylquinazolin-4(3H)-ones, Indian Journal of Chemistry, Section B:Organic Chemistry Including Medicinal Chemistry (2000), 39B(3), 220-222;Desai et al., Quinoline and quinazoline compounds as antitubercularagents, Asian Journal of Chemistry (1998), 10(3), 615-617, Asian Journalof Chemistry; Houghten et al. U.S. Pat. No. 5,783,577 entitled Synthesisof quinazolinone combinatorial libraries and derivatives thereof;Houghten et al., WO 97/10221 entitled Synthesis of quinazolinonelibraries; Couture et al., An expeditious synthesis of 2-aryl- and2-alkylquinazolin-4(3H)-ones, Synthesis (1991), (11), 1009-10; Patersonet al., 1,2,3-Benzotriazin-4-ones and related systems. III. Thermaldecomposition of 3-arylideneamino-1,2,3-benzotriazin-4-ones. Newsynthesis of 2-arylquinazolin-4-one; Breuer et al., U.S. Pat. No.3,753,981 entitled 2-Styryl-4-aminoquinazolines; Matsuoka et al.,Fluorescent whitening agents for synthetic fibers. 41. Fluorescence ofsome quinazolones, Kogyo Kagaku Zasshi (1970), 73(10), 2195-9; Patel etal., Niementowski 4-oxoquinazoline synthesis. I. Modification andmechanism, J. Indian Chem. Soc. (1965), 42(8), 531-5; Mantescu et al.Tritiation of pyrimidines by HTO in the presence of aluminum chloride,J. Labelled Compds. (1965), 1(3), 178-81; Serzhanina et al., Synthesesand transformations of pyrimidine derivatives. XVI. Activity of methylgroups in 2-methylquinazoline derivatives, Zhurnal Organicheskoi Khimii(1965), 1(7), 1303-6; Dhatt et al., 2-Styryl derivatives of4(3)-quinazolones as potential antimalarials and amebicides, CurrentScience (1961), 30, 179-80; Mandasescu et al., Reactivity of methylgroups of benzodiazine. II. Condensation of 2-methylbenzodiazines withaldehydes, Acad. Rep. Populare Romine, Filiala Iasi, Studii CercetariStiint., Chim. (1960), 11, 75-85; Kilroe Smith, Syntheses in thequinazolone series. VI. Synthesis of1,2,3,4-tetrahydro-2-aryl-4-oxoquinazolines, Tetrahedron (1957), 1,38-44; Stephen, Syntheses in the quinazolone series. IV. Conversion ofN-aroylorthanilamides to 2-arylquinazol-4-ones, Journal of the ChemicalSociety (1956) 4420-1; and Bogert, Researches on Quinazolones. XXVI.Synthesis of Some Stilbazoles, Journal of the American Chemical Society(1911), 32, 1654-64. With respect to compounds of Structure 16, seeVieweg et al., Synthesis of new4-oxo-4H-pyrido[3′,2′:4,5]thieno[3,2-d]-1,3-oxazines, Pharmazie (1990),45(10), 731-3.

Representative compounds suitable for the treatment according to thepresent disclosure along with their IC50 values are disclosed in thefollowing Table:

Structure Class ID MOLSTRUCTURE MW IC50 1,1a AB00093511

286.7201 0.079 AB00093467

282.3016 0.082 AB00093558

266.3022 0.1 AB00084000

291.1387 0.062 AB00027137

464.5037 0.13 AB00101507

300.7036 0.337 AB00092734

256.3285 0.382 AB00093094

306.3891 0.788 AB00093093

274.7742 4.062 AB00101695

250.3028 0.579 AB00440877

294.3127 0.639 AB00461411

266.3022 0.968 2 AB00097765

324.4044 0.062 AB00079697

266.3674 0.142 AB00101290

325.4356 0.144 AB00097765

324.4044 0.062 AB00074195

252.3403 0.122 AB00095939

232.3499 0.133 AB00546606

256.3285 0.598 AB00613917

286.355  0.966 AB00546194

274.3439 1.396 3 AB00101018

320.3946 3.172 AB00101133

351.365  3.7 AB00100961

340.8125 3.967 4 AB00443206

378.3598 0.123 AB00171904

453.4614 0.159 AB00174102

509.5697 0.345 5, 5a AB00093745

279.1598 0.251 AB00093742

218.2542 0.382 AB00315863

284.3827 0.65 AB00475708

274.4091 1.926 AB00428616

321.8092 2.412 AB00371839

251.2655 0.125 AB00421150

260.382  12.577 6 AB00011625

432.9262 0.111 AB00012207

452.5985 0.155 AB00003451

464.4885 0.156 AB00547004

441.5125 0.155 AB00317535

398.5305 0.328 AB00542926

482.3591 0.393 7 AB00052939

298.2981 1.725 AB00431689

312.3252 4.524 AB00390364

312.2816 22.698 8 AB00121550

438.5278 0.545 AB00120582

444.9622 145.666 AB00089877

398.5061 1312.209 9 AB00083765

254.3098 3.719 AB00534272

258.3632 5.975 AB00083422

228.2715 3651.481 10, 10a AB00105762

256.2821 0.18 AB00616066

266.3458 0.361 AB00022459

294.4   0.065 AB00013805

338.4536 0.067 AB00616088

314.3004 0.114 AB00329861

268.3181 0.697 11 AB00092070

307.8013 0.775 AB00092117

307.8013 3.403 AB00091724

342.2464 5.186 12 AB00087011

273.7214 0.213 AB00101723

317.7313 0.213 AB00102118

283.3299 0.235 13 AB00548181

342.0241 0.062 AB00528862

279.1598 0.094 14 AB00614173

300.3385 0.104 15 AB00097657

264.2862 0.064 16 AB00079098

284.3391 0.067

Non-limiting examples of neurodegenerative diseases to be treatedaccording to this disclosure are Alzheimer, Parkinson, Amyotrophiclateral sclerosis, Spinal Muscular Atrophy, Brain traumatic injury andassociated neurodegeneration, vascular dementia, Huntington disease andmemory and learning deficit (ADHD, mental retardation).

The following is a description of the assay according to the presentdisclosure.

SH-5YSY human neuroblastoma cell line was obtained from the AmericanTissue Culture collection (ATCC). The cells were expanded and frozen forlong term storage. A commercially available expression vector containingthe NF-kB promoter enhancer region driving the firefly lucipherase geneexpression (see FIG. 1) was obtained. This plasmid was designed fortransient expression studies and was devoid of any antibiotic-resistanceconferring gene. A second plasmid containing the gene conferringresistance to blasticidin was also used. A dual transfection approach toobtain stable cell lines was employed. Prior to transfection,blasticidin sensitivity of the cell line was performed (See FIG. 2). Itwas determined that SH-5YSY cells were sensitive to the antibiotictoxicity and that 3 μg/ml caused total cell death (FIG. 2). Afterexpanding and purifying adequate quantities of these plasmids, SH-5YSYcells were co-transfected with the above two plasmids and the clonalselection of the transfected cells in the presence of blasticidinproceeded. Several clones were identified that were both resistant toblasticidin and expressed the firefly luciferase upon exposure to TNF-α,a known inducer of NF-kB (See FIG. 3). After several assessments, aclone, C1, which expressed high levels of stimulated-luciferaseexpression and maintained it over time (see FIG. 3) was identified. Todate, this clone has been in culture for over 37 passages withoutsignificant decline of the gene of interest induction. The optimalconditions for this assay to be carried out in a high throughput settinghas been determined. Initially, the optimal number of cells needed wasassessed. In FIG. 4 it is shown the effect of using a range of cellsfrom 10,000 to 40,000. The data indicates that a sufficient dynamicrange will be available when using 20,000 cells/well. The assays hashave been implemented using a similar cell density for HTS analysiswithout excessive problems. This cell line allows growing large scalequantities of cells fairly easily.

Successively, the optimal time of incubation and media requirements wereassessed. The data indicates that after a 24 hours settling time,exposure of the cells for 24 hours to positive controls such as TNF-αwill allow for strong induction of the firefly liciferase (data notshown). These conditions are relatively affordable in HTS settings.Finally, since during robotic handling the plates containing the cellsspend a significant amount of time outside the incubator and sincephenol red could interfere with the reagents in the luciferase activityassay kit, the effect of using a phenol red free HEPES-buffered mediawas assessed. As shown in FIG. 5 there is a very little effect of phenolred free, HEPES-buffered media on the assay response and dynamic range.Therefore, the assessment is that it is possible to run this assay usingthese more forgiving media. Since the compounds contained in the mostcommonly used libraries are dissolved in DMSO, the effect of DMSO onboth luciferase expression in response to TNF-α and C1 clone survivalduring assay simulations was characterized. The effect of increasingconcentration of DMSO ranging from 0.05% through 0.5% on TNF-α inductionof luciferase (black line and black scale) is shown in FIG. 6. Also theeffect of DMSO on baseline expression of luciferase in the C1 clone isshown in FIG. 6 (gray line and gray scale). In FIG. 7, it is shown theeffect of DMSO on cell survival in the same conditions described in FIG.6. In these experimental settings cells, settled for 24 hours, and thenwere incubated for an additional 24 hours with DMSO at the indicatedconcentrations. At the end of the incubation phase cell viability wasassessed using the commercially available kit Cell Titer Glo™ followingthe manufacturer instructions (Perkin Elmer). Results reported in FIG. 7indicate that DMSO up to 0.5% does not affect either cell viability orTNF-α induction of the luciferase reporter. Finally, the effect ofdifferent concentrations of TNF-α was studied in the clone C1.Increasing the concentration of TNF-α from 0.625 up to 40 ng/ml caused alinear increase of the luciferase expression in C1 cells. Furtherincrease in TNF concentration did not cause any additional increase ofluciferase activity. This indicates that during the screen thatcompounds able to activate the promoter with different efficiency willbe easily picked up by our detection system (FIG. 8).

Essential to development of an assay for high throughput screening isthe assessment of Z′ values. This statistical parameter assesses thepossibility that data obtained in a single well are statisticallysignificant. In general, assays can generate negative and positive Z′values. Negative Z′ values indicate a very unpredictable assay. Z′values comprised between 0 and 0.5 indicates an assay with a certaindegree of uncertainty. Finally Z′ values above 0.5 indicate very robustassays. Z′ values above 0.7 both in canonical quadrant Z′ plates arraysand in scrambled Z′ plates arrays have been produced consistently inlaboratory settings (see FIG. 9). This indicates that the assay is veryrobust.

This assay has been adapted to 96-, 384-, and 1536-well plate format.Higher density formats are also possible. A high throughput screeningcampaign of 300,000 compounds in 1536-well format has been conductedsuccessfully. FIG. 10 depicts the Z′ values for the 160 plates used.

In addition, the data shows that a number of compounds are selectivelyeffective in neurons, that they increase MnSOD activity and that theyare neuroprotective on two different neurodegenerative in vitroparadigms. In particular, the data indicates activation of NF-kB p65 inastrocytes by 8 of the 18 compounds, shown in FIG. 11. Also shown areadditional compounds that were inactive in astrocytes, but are able toincrease NF-kB-driven MnSOD activity in primary cortical neurons inculture. MnSOD is a key enzyme in inactivating ROS, the end point ofalmost all neurodegenerative insults. This enzyme is under the directcontrol of NF-kB as shown in the literature. Therefore, increased MnSODactivity can be a reporter for NF-kB activation and a reliable indicatorof neuroprotective activity. Compounds SRI 22772, 22774, 22773, 22780,22782, 22817, 22820, 22864 (see FIGS. 11 and 12) were able to activatein an expression-dependent manner NF-kB p65 in primary astrocytes. Ithas now been found that compounds deemed inactive in astrocytes, areable to increase NF-kB-induced MnSOD activity and expression in primaryneurons. Compounds SRI 22781, 22818, 22776, 22819 were all active inneurons but not in astrocytes. Only SRI22777 thus far is inactive inthese assays.

All compounds belonging to the group of active in astrocytes tested thusfar in the MnSOD activity assay increased MnSOD activity in neurons. Infact, compounds 22817, 22780, 22782, 22820 were able to induce a largeincrease of MnSOD activity in primary neurons. On the other hand,compounds SRI 22781, 22818, 22776, 22819 belonging to the group ofcompounds inactive in astrocytes, were able to increase NF-kB-inducedMnSOD activity in primary neurons (see FIGS. 16 and 20B). The discoveryof these neuron-selective compounds suggests the possibility thatcompounds exist that activate NF-kB p65 in neurons but not inastrocytes. This feature could be important since activation of NF-kB inastrocytes could have unwanted effects. However, it needs to be pointedout that the final effect of NF-kB activation in glial cells is unknownat this stage and does not represent a disqualifying factor for activecompound selection. Regardless, having compounds active in neurons whichare not active in astrocytes can be a very important and interestingaspect of our research. In FIGS. 16 and 20B, it is shown the effect ofthe four above mentioned compounds on MNSOD expression in primaryneurons. At the concentration tested, the compounds were as potent as ormore potent than TNF-α (an extremely powerful inducer of MnSOD).Stimulation of MnSOD activity by the compounds exceeded 11 folds of thebasal enzyme activity.

Neuron selective compounds 22781 and 22818 show neuroprotective featuresin vitro. These two compounds belonging to the active in neurons butinactive in astrocytes category cause protection of neurons in differentparadigm toxicity experiments. These two compounds, which are the onlytwo tested, selectively increase NF-kB-driven MnSOD activity in neuronsbut do not activate NF-kB p65 in astrocytes. In panel A of FIG. 17, itis shown the protective effect of compound 22781 on glutamateexcitotoxicity. Primary neurons pretreated with our compound for 1 hourwere exposed to a toxic concentration of glutamate in the presence ofglycine and in absence of magnesium for 1 hour and then replaced withtheir original culture media containing the compound and incubated foradditional 24 hours in the presence of the compound or vehicle. At theend of the experimental period, the cells were analyzed using standardimage-based or biochemical viability assays. In Panel A, it is shownthat SRI22781 had no direct toxic effect and that glutamate causedsignificant cell death. However, cells pretreated with SRI22781 wereprotected from glutamate adverse effects in a statistically significantmanner. In panel B, the effect of compound 22818 on NMDA inducedneurotoxicity is shown. Primary neurons were exposed to the compound SRI22818 at 3 μM for 36 hours prior to NMDA toxicity and were presentduring the following incubation prior to quantification of cellviability. NMDA exposure lasted 1 hour. Compound 22818 did not affectgeneral viability but was able to decrease NMDA toxicity by 50% in astatistically significant manner. Finally, compound 22818 also wastested for its effect on β-Amyloid toxicity. β-amyloid (1-42) at 20 μMwas used in these experiments in its fibrillar form. Fibrillar amyloidwas obtained accordingly to manufacturer instructions by preincubatingthe agent at 370° C. in an ad hoc saline solution for 48 hours prior tothe experiments. Fibrillar amyloid caused significant neuronal deathover the 24 hour incubation, as quantified via multiple image-based andbiochemical assays. Neurons were pre-exposed to 3 μM SRI22818, orvehicle, for 1 hour prior to exposure to amyloid and present throughoutthe incubation with the toxin. SRI2281 completely prevented fibrillaramyloid toxic effect, a very promising result.

Formulations

Compounds of the present disclosure can be administered by anyconventional means available for use in conjunction withpharmaceuticals, either as individual therapeutic agents or in acombination of therapeutic agents. They can be administered alone, butgenerally administered with a pharmaceutical carrier selected on thebasis of the chosen route of administration and standard pharmaceuticalpractice. The compounds can also be administered in conjunction withother therapeutic agents if desired.

The pharmaceutically acceptable carriers described herein, for example,vehicles, adjuvants, excipients, or diluents, are well-known to thosewho are skilled in the art. Typically, the pharmaceutically acceptablecarrier is chemically inert to the active compounds and has nodetrimental side effects or toxicity under the conditions of use. Thepharmaceutically acceptable carriers can include polymers and polymermatrices.

The compounds of this disclosure can be administered by any conventionalmethod available for use in conjunction with pharmaceuticals, either asindividual therapeutic agents or in a combination of therapeutic agents.

The dosage administered will, of course, vary depending upon knownfactors, such as the pharmacodynamic characteristics of the particularagent and its mode and route of administration; the age, health andweight of the recipient; the nature and extent of the symptoms; the kindof concurrent treatment; the frequency of treatment; and the effectdesired. A daily dosage of active ingredient can be expected to be about0.001 to 1000 milligrams (mg) per kilogram (kg) of body weight, with thepreferred dose being 0.1 to about 30 mg/kg.

Dosage forms (compositions suitable for administration) typicallycontain from about 1 mg to about 500 mg of active ingredient per unit.In these pharmaceutical compositions, the active ingredient willordinarily be present in an amount of about 0.5-95% weight based on thetotal weight of the composition.

The active ingredient can be administered orally in solid dosage forms,such as capsules, tablets, and powders, or in liquid dosage forms, suchas elixirs, syrups and suspensions. It can also be administeredparenterally, in sterile liquid dosage forms. The active ingredient canalso be administered intranasally (nose drops) or by inhalation of adrug powder mist. Other dosage forms are potentially possible such asadministration transdermally, via patch mechanism or ointment.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachets,tablets, lozenges, and troches, each containing a predetermined amountof the active ingredient, as solids or granules; (c) powders; (d)suspensions in an appropriate liquid; (e) suitable emulsions; and longacting or delayed release formulations. Liquid formulations may includediluents, such as water and alcohols, for example, ethanol, benzylalcohol, propylene glycol, glycerin, and the polyethylene alcohols,either with or without the addition of a pharmaceutically acceptablesurfactant, suspending agent, or emulsifying agent. Capsule forms can beof the ordinary hard- or soft-shelled gelatin type containing, forexample, surfactants, lubricants, and inert fillers, such as lactose,sucrose, calcium phosphate, and corn starch. Tablet forms can includeone or more of the following: lactose, sucrose, mannitol, corn starch,potato starch, alginic acid, microcrystalline cellulose, acacia,gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium,talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid,and other excipients, colorants, diluents, buffering agents,disintegrating agents, moistening agents, preservatives, flavoringagents, and pharmacologically compatible carriers. Lozenge forms cancomprise the active ingredient in a flavor, usually sucrose and acaciaor tragacanth, as well as pastilles comprising the active ingredient inan inert base, such as gelatin and glycerin, or sucrose and acadia,emulsions, and gels containing, in addition to the active ingredient,such carriers as are known in the art.

The compounds of the present disclosure, alone or in combination withother suitable components, can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, and nitrogen. They also may beformulated as pharmaceuticals for non-pressured preparations, such as ina nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The compound can be administered in a physiologically acceptable diluentin a pharmaceutical carrier, such as a sterile liquid or mixture ofliquids, including water, saline, aqueous dextrose and related sugarsolutions, an alcohol, such as ethanol, isopropanol, or hexadecylalcohol, glycols, such as propylene glycol or polyethylene glycol suchas poly(ethyleneglycol) 400, glycerol ketals, such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, an oil, a fatty acid, afatty acid ester or glyceride, or an acetylated fatty acid glyceridewith or without the addition of a pharmaceutically acceptablesurfactant, such as a soap or a detergent, suspending agent, such aspectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters. Suitablesoaps for use in parenteral formulations include fatty alkali metal,ammonium, and triethanolamine salts, and suitable detergents include (a)cationic detergents such as, for example, dimethyldialkylammoniumhalides, and alkylpyridinium halides, (b) anionic detergents such as,for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether,and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergentssuch as, for example, fatty amine oxides, fatty acid alkanolamides, andpolyoxyethylene polypropylene copolymers, (d) amphoteric detergents suchas, for example, alkyl β-aminopropionates, and 2-alkylimidazolinequaternary ammonium salts, and (e) mixtures thereof.

The parenteral formulations typically contain from about 0.5% to about25% by weight of the active ingredient in solution. Suitablepreservatives and buffers can be used in such formulations. In order tominimize or eliminate irritation at the site of injection, suchcompositions may contain one or more nonionic surfactants having ahydrophile-lipophile balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations ranges from about 5% toabout 15% by weight. Suitable surfactants include polyethylene sorbitanfatty acid esters, such as sorbitan monooleate and the high molecularweight adducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol.

Pharmaceutically acceptable excipients are also well-known to those whoare skilled in the art. The choice of excipient will be determined inpart by the particular compound, as well as by the particular methodused to administer the composition. Accordingly, there is a wide varietyof suitable formulations of the pharmaceutical composition of thepresent disclosure. The following methods and excipients are merelyexemplary and are in no way limiting. The pharmaceutically acceptableexcipients preferably do not interfere with the action of the activeingredients and do not cause adverse side-effects. Suitable carriers andexcipients include solvents such as water, alcohol, and propyleneglycol, solid absorbants and diluents, surface active agents, suspendingagent, tableting binders, lubricants, flavors, and coloring agents.

The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tablets.The requirements for effective pharmaceutical carriers for injectablecompositions are well known to those of ordinary skill in the art. SeePharmaceutics and Pharmacy Practice, J.B. Lippincott Co., Philadelphia,Pa., Banker and Chalmers, Eds., 238-250 (1982) and ASHP Handbook onInjectable Drugs, Toissel, 4th ed., 622-630 (1986).

Formulations suitable for topical administration include lozengescomprising the active ingredient in a flavor, usually sucrose and acaciaor tragacanth; pastilles comprising the active ingredient in an inertbase, such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the active ingredient in a suitable liquidcarrier; as well as creams, emulsions, and gels containing, in additionto the active ingredient, such carriers as are known in the art.

Additionally, formulations suitable for rectal administration may bepresented as suppositories by mixing with a variety of bases such asemulsifying bases or water-soluble bases. Formulations suitable forvaginal administration may be presented as pessaries, tampons, creams,gels, pastes, foams, or spray formulas containing, in addition to theactive ingredient, such carriers as are known in the art to beappropriate.

Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field.

The dose administered to an animal, particularly a human, in the contextof the present disclosure should be sufficient to affect a therapeuticresponse in the animal over a reasonable time frame. One skilled in theart will recognize that dosage will depend upon a variety of factorsincluding a condition of the animal, the body weight of the animal, aswell as the severity and stage of the condition being treated.

A suitable dose is that which will result in a concentration of theactive agent in a patient which is known to affect the desired response.The preferred dosage is the amount which results in maximum inhibitionof the condition being treated, without unmanageable side effects.

The size of the dose also will be determined by the route, timing andfrequency of administration as well as the existence, nature, and extendof any adverse side effects that might accompany the administration ofthe compound and the desired physiological effect.

Useful pharmaceutical dosage forms for administration of the compoundsaccording to the present disclosure can be illustrated as follows:

Hard Shell Capsules

A large number of unit capsules are prepared by filling standardtwo-piece hard gelatine capsules each with 100 mg of powdered activeingredient, 150 mg of lactose, 50 mg of cellulose and 6 mg of magnesiumstearate.

Soft Gelatin Capsules

A mixture of active ingredient in a digestible oil such as soybean oil,cottonseed oil or olive oil is prepared and injected by means of apositive displacement pump into molten gelatin to form soft gelatincapsules containing 100 mg of the active ingredient. The capsules arewashed and dried. The active ingredient can be dissolved in a mixture ofpolyethylene glycol, glycerin and sorbitol to prepare a water misciblemedicine mix.

Tablets

A large number of tablets are prepared by conventional procedures sothat the dosage unit was 100 mg of active ingredient, 0.2 mg. ofcolloidal silicon dioxide, 5 mg of magnesium stearate, 275 mg ofmicrocrystalline cellulose, 11 mg. of starch, and 98.8 mg of lactose.Appropriate aqueous and non-aqueous coatings may be applied to increasepalatability, improve elegance and stability or delay absorption.

Solid oral dosage forms may be made by conventional and novel processes.These units are taken orally without water for immediate dissolution anddelivery of the medication. The active ingredient is mixed in a liquidcontaining ingredient such as sugar, gelatin, pectin and sweeteners.These liquids are solidified into solid tablets or caplets by freezedrying and solid state extraction techniques. The drug compounds may becompressed with viscoelastic and thermoelastic sugars and polymers oreffervescent components to produce porous matrices intended forimmediate release, without the need of water.

Long-Acting or Delayed Release Formulations can be made by conventionaland novel processes, which provide for the release of the activecompound over a extended period of time. For example, the delayedrelease formulation can be prepared as an oral dosage form that passesthrough the stomach intact and dissolved in the small intestine or aninjectible formulation that provides for the sustained release of theactive compound into the blood stream over an extended period of time.Moreover, these type of formulations can be, for example, in the form ofan emulsion, suspension, solution, and/or an enteric coated tablet orcapsule.

Moreover, the compounds of the present disclosure can be administered inthe form of nose drops, or metered dose and a nasal or buccal inhaler.The drug is delivered from a nasal solution as a fine mist or from apowder as an aerosol.

The term “comprising” (and its grammatical variations) as used herein isused in the inclusive sense of “having” or “including” and not in theexclusive sense of “consisting only of.” The terms “a” and “the” as usedherein are understood to encompass the plural as well as the singular.

The term “patient” or “subject” means an animal (e.g., cow, horse,sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit, guineapig, etc.) or a mammal, including chimeric and transgenic animals andmammals. In one embodiment, the term “patient” or “subject” means amonkey or a human, most preferably a human. In certain embodiments, thepatient is a human infant, child, adolescent, adult, or geriatricpatient. In a particular embodiment, the patient is a healthyindividual, e.g., an individual not displaying symptoms of memoryimpairment or not suffering from a neurodegenerative disease.

All publications, patents and patent applications cited in thisspecification are herein incorporated by reference, and for any and allpurpose, as if each individual publication, patent or patent applicationwere specifically and individually indicated to be incorporated byreference. In the case of inconsistencies, the present disclosure willprevail.

The foregoing description of the disclosure illustrates and describesthe present disclosure. Additionally, the disclosure shows and describesonly the preferred embodiments but, as mentioned above, it is to beunderstood that the disclosure is capable of use in various othercombinations, modifications, and environments and is capable of changesor modifications within the scope of the concept as expressed herein,commensurate with the above teachings and/or the skill or knowledge ofthe relevant art.

The embodiments described hereinabove are further intended to explainbest modes known of practicing it and to enable others skilled in theart to utilize the disclosure in such, or other, embodiments and withthe various modifications required by the particular applications oruses. Accordingly, the description is not intended to limit it to theform disclosed herein. Also, it is intended that the appended claims beconstrued to include alternative embodiments.

1. (canceled)
 2. A method of treating a patient suffering from a neurodegenerative disease, the method comprising administering to the patient in need thereof a therapeutically effective amount of a compound selected from the group consisting of: a compound of formula (2):

wherein in formula (2): Y is O or S; R₁ is H, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, and substituted or unsubstituted heteroaryl; R₂ is each selected from the group consisting of H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, and substituted or unsubstituted heteroaryl; or R₁, R₂ combine with the N atom to which both groups are covalently linked as to form the morpholin-1-yl group; R₃ is selected from the group consisting of H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, and substituted or unsubstituted heteroaryl; and R₄ is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, and substituted or unsubstituted heteroaryl; and a compound of formula (12):

wherein in formula (12): R₁ represents one or more substituents on the phenyl ring, and each occurrence of R₁ is independently selected from the group consisting of H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heteroaryl, acyl, halogen, hydroxy, alkoxy, and amino or substituted amino, or two adjacent R₁ groups on the phenyl ring combine as to form the —CH₂OCH₂— group; R₂ is selected from the group consisting of H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, and substituted or unsubstituted heteroaryl; R₃ is selected from the group consisting of H, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, and substituted or unsubstituted heteroaryl; and the carbon labeled as β is optionally further substituted with a CH₃ group.
 3. The method of claim 2, wherein in formula (2) Y is S.
 4. The method of claim 2, wherein in formula (2) R₃ is H.
 5. The method of claim 2, wherein in formula (2) R₁ is H.
 6. The method of claim 2, wherein in formula (2) R₂ is selected from the group consisting of H, substituted or unsubstituted alkyl, and substituted or unsubstituted aryl, or R₁, R₂ combine with the N atom to which both groups are covalently linked as to form the morpholin-1-yl group.
 7. The method of claim 2, wherein the compound of formula (2) is selected from the group consisting of:


8. The method of claim 2, wherein in formula (12) at least one occurrence of R₁ is selected from the group consisting of H, halogen and alkoxy, or two adjacent R₁ groups on the phenyl ring combine as to form the —CH₂OCH₂— group.
 9. The method of claim 2, wherein R₂ is H.
 10. The method of claim 2, wherein R₃ is substituted or unsubstituted aryl.
 11. The method of claim 2, wherein the carbon labeled as β is not further substituted with a methyl group.
 12. The method of claim 2, wherein the carbon labeled as β is further substituted with a methyl group.
 13. The method of claim 2, wherein the compound of formula (12) is selected from the group consisting of:


14. The method of claim 2, wherein the neurodegenerative disease is at least one selected from the group consisting of Alzheimer's Disease, Parkinson's Disease, amyotrophic lateral sclerosis, spinal muscular atrophy, brain traumatic injury and associated neurodegeneration, vascular dementia, Huntington's Disease, and memory and learning deficit.
 15. The method of claim 2, wherein the compound is administered to the patient as part of a pharmaceutical composition further comprising at least one pharmaceutically acceptable excipient.
 16. The method of claim 2, wherein the compound is administered to the patient by at least one route selected from the group consisting of oral, inhalation, nasal, buccal, parenteral, topical, rectal, and vaginal routes.
 17. The method of claim 2, wherein the patient is a mammal.
 18. The method of claim 17, wherein the patient is human. 